In Vitro Reversion of Amphotericin B Resistance in Leishmania donovani by Poloxamer 188

Abstract

A micellar formulation of amphotericin B (AmB) solubilized with poloxamer 188 was evaluated against an AmB Leishmania donovani-resistant line. A concave isobologram showed a synergistic effect of this association against promastigotes. This result was confirmed with amastigotes since the 50% effective concentration of the new formulation was 100 times less than that of the control AmB formulation.The use of amphotericin B (AmB) for the treatment of visceral leishmaniasis (VL) is increasing as a consequence of the worldwide spread of resistance to the first-line pentavalent antimonials (7) and the improvement of the therapeutic index of AmB in the commercialized lipidic formulations (AmBisome, Amphocil, and Albecet). Clinical trials have indicated that these formulations decrease the toxicity of the drug and that they are also more active against this parasite (3,4,5,8,9,10,17,22,24).Since AmB is increasingly used, the risk of the appearance of clinical resistance could increase. In anticipation of this fact, a line of AmB-resistant (AmB r ) Leishmania donovani promastigotes was established by stepwise drug pressure (16), and their biological properties were compared with those of the wild-type (WT) parent strain. Ergosterol, the main target of AmB in fungi, was present in the membranes of WT strains but was not found in the membranes of the AmB-resistant line of isolates (16). This modification was accompanied by an increase in membrane fragility and fluidity. The AmB-resistant promastigotes were infective for macrophages in vitro, but their virulence was considerably decreased in vivo. Strategies that can be used to overcome the resistance should be investigated.In a previous work (11), we studied the physicochemical properties of a formulation of AmB solubilized with poloxamer 188. Poloxamers are water-soluble, nonionic, triblock copolymeric surfactants of poly(ethylene oxide) and poly(propylene oxide) (20). Among them, poloxamer 188 was approved by the Food and Drug Administration as a safe ingredient for injections (1), and it is reported in the National Formulary as a pharmaceutical ingredient (23). These formulations were less toxic to red blood cells, macrophages, and renal cells and were also less toxic in vivo in noninfected mice (M. S. Espuelas, P. Legrand, P. Loiseau, C. Bories, C. Gamazo, J.-P. Devissaguet, M. J. Renedo, and J. M. Irache, Proc. 1st World Meet. APGI/ APV on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technol. p. [569][570] 1998).The sizes of AmB aggregates decreased and the degree of AmB self-aggregation increased with the poloxamer concentration used to solubilize the drug. As Mullen et al. (19) reported a correlation between the degree of AmB aggregation in the lipidic formulations and their antileishmanial activities, in the present study, the antileishmanial activity of the micellar formulation of AmB solubilized with poloxamer 188 at two different concentrations was assessed in vitro against both WT and AmB r L. donovani DD8 (strain MHOM/IN/80/DD8) promastigotes and intram...

Toxicity and Antileishmanial Activity of a New StableLipid Suspension of AmphotericinB

“…It is possible that some formulations could improve the efficacy of AMB by facilitating its insertion into the modified parasite membrane. These results were not as spectacular as those obtained by Espuelas et al (13), in which mixed micelles of AMB with the hydrophilic surfactant poloxamer 188 were able to completely reverse the resistance of the DD8 strain in vitro.…”

Chemistry, cytotoxicity and antileishmanial activity of the essential oil from Piper auritum

“…Non-adherent cells were removed by washing with pre-warmed phosphate-buffered saline (PBS). Stationary-phase L. donovani promastigotes were added at a 10:1 parasite/ macrophage ratio and the cultures were incubated for another 24 h. The cell monolayers were washed three times with pre-warmed PBS to remove free parasites and 999 µL of RPMI completed medium and 1 µL of the essential oil dissolved in DMSO, were added in duplicate and incubated for an additional 48 h (Espuelas et al 2000). The cells and parasites were then fixed in absolute methanol, stained with Giemsa and examined under light microscopy.…”

In vitro activity of an essential oil against Leishmania donovani

“…Stationary-phase L. donovani promastigotes were added at a 10:1 parasite/macrophage ratio, and the cultures were incubated for further 24 h. The cell monolayers were washed three times with pre-warmed PBS to remove free parasites. Then, 999 μL of RPMI completed medium and 1 μL of the essential oil dissolved in DMSO, at final concentrations between 1 to 16 μg/mL, were added in duplicate for further 48 h (Espuelas et al, 2000). The cells and parasites were then fixed in absolute methanol, stained with Giemsa, and examined under light microscopy.…”